Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 10(9) CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 10(4) CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 10(6) CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.     

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium

The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature-tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI-4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI-4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many 'housekeeping' genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.     

Enhanced resistance to Salmonella enterica serovar Typhimurium infection in mice after coumarin treatment

Coumarin and its derivatives are naturally occurring substances with multiple biological activities. Here we demonstrate that prophylactic peroral administration of coumarin or 7-hydroxycoumarin (7-OHC) enhances resistance to subsequent lethal Salmonella enterica Serovar Typhimurium infection in mice. 7-OHC decreased bacterial load in liver and spleen, and enhanced phagocytosis and bacterial killing by macrophages when applied in vitro and in vivo. 7-OHC treatment induced significant NO release in peritoneal macrophage cultures. The observed protective effect correlated with the induction of Th1-associated cytokines, such as IL-12, IFN-gamma, and TNF-alpha. These data demonstrate a clear immunomodulatory potential of coumarins which might have important therapeutic implications to enhance resistance to infection.     

Influence of infection route and virulence factors on colonization of solid tumors by Salmonella enterica serovar Typhimurium

Administration of facultative anaerobic bacteria such as Salmonella enterica serovar Typhimurium as anticancer treatment holds a great therapeutic potential. Here, we tested different routes of application of S. typhimurium with regard to tumor colonization and therapeutic efficacy. No differences between intravenous and intraperitoneal infection were observed, often leading to a complete tumor clearance. In contrast, after oral application, tumor colonization was inefficient and delayed. No therapeutic effect was observed under such conditions. We also showed that tumor invasion and colonization were independent of functional Salmonella pathogenicity island (SPI) 1 and SPI 2. Furthermore, tumor invasion and colonization did not require bacterial motility or chemotactic responsiveness. The distribution of the bacteria within the tumor was independent of such functions.     

Membrane topology of the ZntB efflux system of Salmonella enterica serovar Typhimurium

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.     

SopD acts cooperatively with SopB during Salmonella enterica serovar Typhimurium invasion

The intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. typhimurium), causes disease in a variety of hosts. To invade and replicate in host cells, these bacteria subvert host molecular machinery using bacterial proteins, called effectors, which they translocate into host cells using specialized protein delivery systems. One of these effectors, SopD, contributes to gastroenteritis, systemic virulence and persistence of S. typhimurium in animal models of infection. Recently, SopD has been implicated in invasion of polarized epithelial cells and here we investigate the features of SopD-mediated invasion. We show that SopD plays a role in membrane fission and macropinosome formation during S. typhimurium invasion, events previously shown to be mediated by the SopB effector. We further demonstrate that SopD acts cooperatively with SopB to promote these events during invasion. Using live cell imaging we show that a SopD-GFP fusion does not localize to HeLa cell cytosol as previously described, but instead is membrane associated. Upon S. typhimurium infection of these cells, SopD-GFP is recruited to the invasion site, and this recruitment required the phosphatase activity of SopB. Our findings demonstrate a role for SopD in manipulation of host-cell membrane during S. typhimurium invasion and reveal the nature of its cooperative action with SopB.     

Bistability in myo-inositol utilization by Salmonella enterica serovar Typhimurium

The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.     

Peptidase N encoded by Salmonella enterica serovar Typhimurium modulates systemic infection in mice

The cytosolic protein degradation pathway, involving ATP-dependent proteases and ATP-independent peptidases, is important for modulating several cellular responses. The involvement of pathogen-encoded ATP-dependent proteases is well established during infection. However, the roles of ATP-independent peptidases in this process are not well studied. The functional role of Peptidase N (PepN), an ATP-independent enzyme belonging to the M1 family, during systemic infection of mice by Salmonella enterica serovar Typhimurium (Salmonella typhimurium) was investigated. In a systemic model of infection, the number of CFU of S. typhimurium containing a targeted deletion in peptidase N (DeltapepN), compared with wild type, was significantly higher in the lymph node and spleen. In addition, S. typhimurium replicated in the thymus and greatly reduced the number of immature CD4(+)CD8(+) thymocytes in a dose- and time-dependent manner. Strains lacking or overexpressing pepN were used to show that the reduction in the number of thymocytes, but not lymph node cells, depends on a critical number of CFU. These findings establish a role for PepN in reducing the in vivo CFU of S. typhimurium during systemic infection. The implications of these results, in the context of the roles of proteases and peptidases, during host-pathogen interactions are discussed.     

Transcriptional profile of Salmonella enterica subsp. enterica serovar Weltevreden during alfalfa sprout colonization

Sprouted seeds represent a great risk for infection by human enteric pathogens because of favourable growth conditions for pathogens during their germination. The aim of this study was to identify mechanisms of interactions of Salmonella enterica subsp. enterica Weltevreden with alfalfa sprouts. RNA-seq analysis of S. Weltevreden grown with sprouts in comparison with M9-glucose medium showed that among a total of 4158 annotated coding sequences, 177 genes (4.3%) and 345 genes (8.3%) were transcribed at higher levels with sprouts and in minimal medium respectively. Genes that were higher transcribed with sprouts are coding for proteins involved in mechanisms known to be important for attachment, motility and biofilm formation. Besides gene expression required for phenotypic adaption, genes involved in sulphate acquisition were higher transcribed, suggesting that the surface on alfalfa sprouts may be poor in sulphate. Genes encoding structural and effector proteins of Salmonella pathogenicity island 2, involved in survival within macrophages during infection of animal tissue, were higher transcribed with sprouts possibly as a response to environmental conditions. This study provides insight on additional mechanisms that may be important for pathogen interactions with sprouts.     

Bile-induced curing of the virulence plasmid in Salmonella enterica serovar Typhimurium

Exposure to bile induces curing of the virulence plasmid in Salmonella enterica serovar Typhimurium (pSLT). Disruption of the ccdB gene increases pSLT curing, both spontaneous and induced by bile, suggesting that the pSLT ccdAB genes may encode a homolog of the CcdAB addiction module previously described in the F sex factor. Unlike the F sex factor, synthesis of pSLT-encoded pili does not confer bile sensitivity. These observations may provide insights into the evolution of virulence plasmids in Salmonella subspecies I, as well as the causes of virulence plasmid loss in other Salmonella subspecies.     

Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S. Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.     

Autophagy Recognizes Intracellular Salmonella enterica serovar Typhimurium in Damaged Vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S.Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.